Method of determining sperm capacitation

ABSTRACT

This invention describes unique patterns of distribution of ganglioside G M1  in non-capacitated sperm and demonstrates that the pattern of distribution of G M1  undergoes changes that can be correlated with the process of capacitation and/or with acrosomal exocytosis. Accordingly, the present invention discloses a method for determining the ability of sperm to respond to capacitation and/or acrosomal exocytosis stimuli. The method comprises determination of distribution pattern for G M1 . The method can be used for both diagnostic and predictive purposes when assessing male reproductive fitness, and can also be used to assess the effects on sperm of cryoprotective agents and protocols, and contraceptive agents.

This application claims priority to U.S. provisional application No.60/489,443, filed on Jul. 23, 2003, the disclosure of which isincorporated herein by reference.

This work was supported by grant numbers KO1-RR00188, PO1-HD-06274 andRO1-HD-045664 from the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the field of male fertility and morespecifically provides a method for determining sperm quality,particularly with regard to assessing the ability of sperm to undergocapacitation by following the pattern of distribution of G_(M1)ganglioside.

DESCRIPTION OF RELATED ART

Ejaculated sperm are not immediately able to fertilize an egg. Rather,they must undergo a process of functional maturation known as“capacitation” (Austin, 1952, Nature, U.S.A., 170:326; Chang, 1951,Nature, U.S.A., 168:697–698). “Capacitation” is generally regarded to bea process that results in the acquisition of hyperactivated motility,and the acquisition of the ability to undergo acrosomal exocytosis.Capacitation results in two specific changes in sperm function. First,the sperm head acquires the ability to undergo acrosomal exocytosis inresponse to physiological ligands such as zona pellucida proteins orprogesterone. Second, the flagellum of the sperm acquires a“hyperactivated” pattern of motility. Although some stimuli forcapacitation are species specific, several stimuli for capacitation arecommon between species. These include the presence of bicarbonate andcalcium ions, and the presence of reagents that can act as sterolacceptors thereby mediating the efflux (removal) of sterols from thesperm plasma membrane (e.g. serum albumin, cyclodextrin, high densitylipoproteins (HDL), etc.). Currently there are no sensitive and simplemarkers for capacitation that can be used in a clinical setting. Forexample, the appearance of protein tyrosine phosphorylation eventsduring the process of capacitation has been described in many species.However, visualization of these events using polyacrylamide gelelectrophoresis and immunoblotting can take upwards of 48 hours toperform, making it ill-suited for clinical purposes.

Although it has long been known that mammalian spermatozoa possessdifferent regions in their plasma membranes, information about thesedomains is largely descriptive. For example, the plasma membraneoverlying the acrosome is known to be enriched in sterols. Functionally,it is known that sperm cannot fertilize an egg until sterols have beenremoved from the sperm plasma membrane by extracellular acceptors in theprocess of capacitation. However, it is unclear how other lipids areorganized in the sperm plasma membrane and if the organization of theselipids might reflect changes associated with the process ofcapacitation. Accordingly, to address various issues related to malefertility, there is a need to understand lipid patterns duringcapacitation.

SUMMARY OF THE INVENTION

In the present invention, we demonstrate that mammalian sperm possesslipid rafts enriched in sterols and the ganglioside G_(M1). These mayboth organize and regulate signaling pathways in somatic cells. Theselipids are segregated from other regions of the plasma membrane having ahigher content of phospholipids

In addition, we present evidence that identifiable and reproducibledifferences in the pattern of distribution of G_(M1) in these rafts canbe correlated with the presence of specific stimuli known to be involvedin triggering of the process of capacitation and/or acrosomal exocytosis(the “acrosome reaction”).

Further, it was also observed that the pattern of G_(M1) distributionobserved was dependent upon the fixation conditions used. Thus, amongstthe fixation conditions studied, it was observed that using 4%paraformaldehyde with 0.1% glutaraldehyde and 1 mM CaCl₂ resulted in areproducible pattern of G_(M1) distribution that was closest to thedistribution pattern seen in living sperm regardless of whether thesperm were incubated under capacitating or non-capacitating conditions.The use of 0.004% paraformaldehyde as a fixative produces different butreproducible patterns of G_(M1) distribution for sperm incubated undercapacitating and/or acrosomal exocytosis inducing conditions. Forexample, we have observed that in living mouse sperm prior tocapacitation, G_(M1) is localized within the sperm head to the plasmamembrane overlying the acrosome. G_(M1) has also been observed in themid-piece and principal piece of the flagellum. After incubating spermwith reagents (singly or in combination) that stimulate capacitationand/or acrosomal exocytosis, a reproducible and stimulus-specific changein the pattern of distribution of G_(M1) was observed. For example,after incubation of sperm with stimuli for capacitation and acrosomalexocytosis, in both living sperm and sperm fixed with 4%paraformaldehyde, 0.1% glutaraldehyde and 1 mM CaCl₂, GM1 staining inthe area of the apical acrosome was seen. Other patterns of GM1 stainingwere seen under other fixation conditions. For example, in sperm fixedwith 0.004% paraformaldehyde, GM1 staining was seen in thepost-acrosomal plasma membrane. In sperm exposed to bicarbonate,staining was observed in the apical acrosome and post-acrosomal region.In sperm exposed to reagents mediating sterol efflux (such as2-hydroxypropyl)-β-cyclodextrin) a signal in the plasma membraneoverlying the acrosome as well as in the post-acrosomal region wasobserved. In sperm incubated with bicarbonate and mediators of sterolefflux (2-hydroxypropyl)-β-cyclodextrin) and then reagents which induceacrosomal exocytosis (e.g. progesterone), a signal over the apicalacrosome alone was observed.

A distinct pattern of G_(M1) distribution was also seen in all otherspecies examined namely, boar, stallion, human, and dog. These spermhave distinct patterns of localization and redistribution of G_(M1),with the redistribution patterns correlating with specific stimuli forcapacitation and acrosomal exocytosis. The pattern observed is alsodependent upon the fixative used. Based on the observations that G_(M1)is found in the plasma membrane, that it displays a highly reproduciblepattern of localization and that it redistributes upon incubation underspecific conditions (such as after exposure to capacitating stimuli),several applications are described herein.

In one embodiment a pattern of normal distribution of G_(M1) can bedetermined in non-capacitated sperm under living or defined fixationconditions. While various fixation conditions can be used fordetermination of G_(M1) distribution patterns, in a preferredembodiment, fixation conditions are identified which show a distinct andreproducible staining pattern. The distribution pattern of G_(M1) innon-capacitated sperm is designated to be the normal (or control)pattern.

The change in the distribution pattern of G_(M1) in response tocapacitating stimuli and/or stimuli inducing acrosomal exocytosis istermed herein as “redistribution”. This is distinct from a change in theGM1 pattern observed when sperms are fixed with different fixatives.This latter phenomenon is termed as “fixative induced movements” of GM1.In one embodiment of the invention, a change in the distribution patternof GM1 (redistribution) in response to stimuli inducing capacitationand/or acrosomal exocytosis under different fixation conditions can bedocumented. This establishes control patterns for G_(M1) redistributionin response to these stimuli or fixation conditions. Theseredistribution patterns can be used as controls for assessing factorsaffecting capacitation.

In another embodiment, the pattern of G_(M1) distribution innon-capacitated sperm and its ability to undergo redistribution inresponse to specific stimuli for capacitation when compared with anormal population is used as a diagnostic tool for male infertility.

In another embodiment, the pattern of G_(M1) distribution in capacitatedsperm and its ability to undergo redistribution associated withacrosomal exocytosis when compared with a normal population is used as adiagnostic tool for male infertility. The information obtained from thecapacitation and/or acrosomal exocytosis abilities of sperm in a testsample can be useful for clinicians to identify suitable approaches inassisted reproduction methods.

In another embodiment, the pattern of G_(M1) distribution innon-capacitated sperm in a test sample from an individual and theredistribution of the G_(M1) pattern associated with capacitation andacrosomal exocytosis when compared with a normal sperm sample is used asa predictive tool for evaluating future breeding soundness of theindividual.

In another embodiment, the pattern of G_(M1) localization and/or itsredistribution associated with capacitation and/or acrosomal exocytosisis used as a marker to evaluate the effect on sperm of potential semenextenders and cryopreservation agents/protocols.

In another embodiment, the pattern of G_(M1) localization and/or itsredistribution associated with capacitation and/or acrosomal exocytosisis used as a marker to evaluate potential male contraceptives.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation of the localization of G_(M1) in the head ofa living, motile, murine spermatozoon. The G_(M1) is seen localized tothe plasma membrane overlying the acrosome, and is specifically excludedfrom the post-acrosomal plasma membrane.

FIGS. 2A–C are representations of the localization of sterols, caveolin,and G_(M1) in fixed murine sperm. Filipin was used to visualize sterolsby its inherent auto-fluorescent properties (A). Caveolin-1 waslocalized via indirect immuno-fluorescence (B). G_(M1) was localizedusing a fluorescence-labeled b subunit of cholera toxin (C). This cell,incubated under non-capacitating conditions, was fixed using 0.004%paraformaldehyde, showing localization of G_(M1) to the post-acrosomalplasma membrane under this fixation condition

FIGS. 3A–3C are representations of the various patterns of localizationof G_(M1), as visualized with Cholera toxin b (CTb), in murine spermincubated under non-capacitating and capacitating conditions. Thesepatterns are seen when sperms are fixed with 0.004% paraformaldehyde.Two localization patterns are shown grouped together as pattern “B.”This is because the increase in intensity of labeling seen at the edgesof the sub-acrosomal ring were often seen in the same cells having ashift in CTb labeling along the edge of the apical acrosome (Arrowsindicate the sub-acrosomal ring in pattern A and the increase inintensity seen in pattern B.). Pattern “C” was defined on the basis of asimilar intensity of fluorescence between the acrosomal andpost-acrosomal domains. Schematics at the top of the figure are shownwith inverted light intensities.

FIG. 4 is a graphical representation of the relative distribution ofpatterns seen in FIG. 3, when murine sperm are incubated under differentconditions. The figure shows the mean percentages of sperm havingpatterns A, B and C with the associated standard deviations. Betweenfour to eight experiments were performed for each condition, and atleast 100 cells were counted per condition per experiment.

FIGS. 5A and B are representations of the pattern of G_(M1) localizationseen in live murine sperm after incubation under capacitatingconditions, and exposure to a reagent which induces acrosomal exocytosis(e.g. progesterone). A similar pattern is observed with cells fixed with4.0% paraformaldehyde, 0.1% glutaraldehyde, and 1 mM CaCl₂.

FIG. 6 is a representation of several patterns of G_(M1) localizationseen in a normal sample of non-capacitated bull sperm

FIG. 7 is a representation of two patterns of G_(M1) localization seenin a normal sample of non-capacitated dog sperm.

FIGS. 8A and 8B are representations of G_(M1) localization in horsesperm. In 8A, two sperm are shown that represent normal patterns. Theone in the upper right hand corner shows G_(M1) staining predominantlyin the upper half of the sperm head, while the one in the lower leftcorner shows staining in the lower half of the sperm head. In 8B, asperm is shown which shows distinct G_(M1) localization around anabnormality (either a proximal droplet or a “pseudo-droplet”).Interestingly, G_(M1) localization in this particular sperm head alsodeviates from the normal patterns by appearing as a covering over theentire surface.

DETAILED DESCRIPTION OF THE INVENTION

The term “GM₁ staining” or “staining of GM₁” or related terms as usedherein means the staining seen is cells due to the binding of labeledaffinity molecules to GM₁, For example, when fluorescent tagged Choleratoxin b subunit is used for localization of GM₁, the signal or stainingis from the Cholera toxin b subunit but is indicative of the location ofGM₁. The terms “signal” and “staining” are used interchangeably.

The term “capacitated” sperm refers to sperm which have been incubatedunder conditions which promote the process of capacitation.Specifically, as is known in the art, this requires the presence ofbicarbonate and calcium ions in the medium, and the presence of a sterolacceptor such as serum albumin or a cyclodextrin. Capacitated sperm haveacquired the ability to undergo acrosomal exocytosis and have acquired ahyperactivated pattern of motility. Consequently, the term“non-capacitated” sperm refers to sperm which are incubated in theabsence of the above listed stimuli for capacitation. Such sperm cannotundergo acrosomal exocytosis induced by a physiological ligand such asthe zona pellucida, solubilized proteins from the zona pellucida, orprogesterone. In addition, sperm incubated under non-capacitatingconditions also will not demonstrate hyperactivated motility.

The present invention provides a method for assessing the ability ofspermatozoa to undergo capacitation and to evaluate the process ofcapacitation. The method is based on the novel observation that a uniquepattern of distribution of the ganglioside G_(M1) is seen in both livingand fixed, non-capacitated sperm, and that this pattern changes in areproducible manner when the sperm are incubated with different externalstimuli mimicking the events during capacitation and/or acrosomalexocytosis. Since changes in the pattern of distribution of G_(M1) werefound to be dependent upon the fixation conditions, based on theteachings and examples provided herein, fixation conditions providingthe most distinction between non-capacitated sperm and capacitated spermwith or without acrosomal exocytosis can be determined for differentspecies. Because of different patterns of GM1 generated under differentfixation conditions, it is preferable to run control samples under samefixation conditions as the test samples. In a more preferred embodiment,the control samples are run in parallel.

Reproducible changes in the pattern of GM1 localization can be observedin sperm that are first incubated with different external stimuli, andthen are fixed under defined conditions. Manipulating either/both theincubation conditions and the fixation conditions, can therefore give aninvestigator different types of complementary information. For example,to evaluate if a given population of mouse sperm has normal organizationof membrane sub-domains under non-capacitating conditions and/or inresponse to stimuli for capacitation and exocytosis, one could determineGM1 staining in living cells or in cells fixed with 4% paraformaldehyde,0.1% glutaraldehyde, and 1 mM CaCl₂. While not intending to be bound byany particular theory, it is considered that under these fixationconditions, the lipids are locked in place as they are in the livingsperm. In another embodiment, to evaluate the percent of sperm that arecapable of responding to stimuli for capacitation thereby providingpredictive/diagnostic information about the ability of the sperm toundergo specific stages of capacitation and be competent to fertilize anegg, the cells could be fixed with 0.004% paraformaldehyde, prior tolabeling to localize G_(M1). Under this fixation condition, we seereproducible patterns of movements of G_(M1) specifically induced inonly those cells that are responding to the stimuli. These patterns canbe compared to “normal” or “control” patterns of G_(M1) distribution insperm from fertile males incubated and fixed under the same conditions.Comparison of the sperm in the test sample with the normal sperm wouldthen be the basis of declaring the sperm normal or abnormal.

For visualization of G_(M1) in living sperm, the spermatozoa aretypically washed with a standard base medium (e.g. Modified Whitten'smedium, or other media which support the function of sperm of differentspecies) and incubated with an affinity molecule for G_(M1) which has adetectable moiety on it. Since G_(M1) has extracellular sugar residueswhich can serve as an epitope, it can be visualized without having tofix and permeabilize the cells. However, fixation of the cells willresult in better preservation of the specimen, easy visualization(compared to discerning patterns in swimming sperm) and allow longervisualization time. Based on the teachings provided herein,determination of fixation conditions that will produce a reproduciblepattern of distribution of G_(M1) can be done by those skilled in theart. Various fixatives known for histological study of spermatozoa arewithin the purview of those skilled in the art. Suitable fixativesinclude paraformaldehyde, glutaraldehyde, Bouin's fixative, andfixatives comprising sodium cacodylate, calcium chloride, picric acid,tannic acid and like. In a preferred embodiment, paraformaldehy,glutaraldehyde or combinations thereof are used.

In the mouse, when using 0.004% paraformaldehyde as a fixative (FixativeA in Table 1), almost all non-capacitated sperm show a post-acrosomalpattern. After incubation of sperm with bicarbonate alone, approximately40% of the cells show signal over the apical acrosome as well as overthe post-acrosomal plasma membrane. After incubation of the sperm withcyclodextrin, approximately 40% of the cells showed a diffuse patternthroughout the head of the sperm. Note: in the table, Pattern A standsfor “acrosomal,” pattern “D” stands for diffuse, pattern “AA/PA” standsfor “apical acrosome and post-acrosomal” and “PA” stands for“post-acrosomal.” “NC” stands for non-capacitating and “CAP” stands forcapacitating (in the presence of both cyclodextrin and bicarbonate).

When using 1.25% paraformaldehyde, 2.5% glutaraldehyde, 100 mM sodiumcacodylate and 0.5 mM CaCl₂ as a fixative (Fixative B in Table 1),approximately 80% of the sperm incubated under non-capacitatingconditions showed signal over the acrosomal plasma membrane, with theremainder having a diffuse pattern. When incubated in the presence of asterol acceptor, approximately 60% of the sperm exhibit a diffusepattern.

When using 4% paraformaldehyde, 0.1% glutaraldehyde and 1 mM CaCl₂ as afixative (Fixative C in Table 1), approximately 80% of the spermincubated under any of the conditions display signal over the acrosomalplasma membrane. The remainder display a diffuse pattern.

TABLE 1 Pattern Incubation AA/ Fixative Condition A D PA PA A NC  ns*7.3 17.3 72.8 NaHCO₃ Ns 3.2 34.9 61.8 2-OHCD Ns 40.4 28.8 37.9 CAP Ns33.6 26.9 39.6 B NC 75.2 0 0 24.8 NaHCO₃ 49.0 0 0 51.0 2-OHCD 41.8 0 058.2 CAP 37.9 0 0 62.3 C NC 83.7 0 0.2 16.2 NaHCO₃ 78.2 0 0.9 20.82-OHCD 80.5 0 0.2 19.4 CAP 79.6 0 0 20.4

In living normal sperm (unfixed), almost all of the cells show patternA.

During the process of capacitation, sperm are known to respond toexternal stimuli such as bicarbonate and calcium ions, and mediators ofsterol efflux such as 2-hydroxy-propyl cyclodextrin,methyl-β-cyclodextrin, serum albumin, high density lipoprotein,phospholipids vesicles, liposomes etc. An identifiable change in theG_(M1) distribution pattern has been observed when spermatozoa areexposed to these stimuli in vitro.

Unique G_(M1) staining patterns have been observed in mouse, horse,boar, dog and human sperm. In addition, abnormalities in G_(M1)distribution can be seen in the heads of sperm havingmorphologically-obvious flagellar defects. Thus, localization of G_(M1)can be used to indicate defects in sperm membranes not detectable bynormal morphological observations without the G_(M1) staining. Forexample, flagellar defects (such as both proximal and distal cytoplasmicdroplets) may not necessarily indicate abnormal capacitation ability.Visualization of G_(M1) distribution patterns and comparison withcontrols will provide a more accurate determination of the capacitationability.

The distribution pattern of G_(M1) in live or fixed sperm can beobtained by using affinity binding techniques. A molecule havingspecific affinity for the G_(M1) ganglioside can be used. The affinitymolecule can be directly linked to a detectable label (such as afluorophore) or may be detected by a second affinity molecule which hasa detectable label on it. For example, the b subunit of cholera toxin isknown to specifically bind to G_(M1). Therefore, a labeled (such asfluorescent labeled) cholera toxin b subunit can be used to obtain apattern of distribution of G_(M1). Alternatively, a labeled antibody tothe cholera toxin b subunit can be used to visualize the pattern ofG_(M1) staining. The detectable label is such is that it is capable ofproducing a detectable signal. Such labels include a radionuclide, anenzyme, a fluorescent agent or a chromophore. Staining and visualizationof G_(M1) distribution in sperm is carried out by standard techniques.Affinity molecules other than the b subunit of cholera toxin can also beused. These include polyclonal and monoclonal antibodies. Specificantibodies to G_(M1) ganglioside can be generated by routineimmunization protocols, or can be purchased commercially (Matreya, Inc.,State College, Pa.). The antibodies may be raised against G_(M1) or,since gangliosides in general, are known to be weak antigens, specificantibodies to G_(M1) can be generated by using peptide mimics ofrelevant epitopes of the G_(M1) molecule. Identification and generationof peptide mimics is well known to those skilled in the art.

G_(M1) in sperm lipid rafts can be visualized on living, motile sperm,or on sperm that are fixed. We demonstrate that when sperm are fixedunder different conditions, different patterns of localization can beseen. These patterns reflect whether the sperm have responded tocapacitating stimuli in the medium with which they were incubated (forexample, bicarbonate or agents mediating sterol efflux, two of thecritical upstream stimuli for capacitation). In this way, the inventionallows one to track the percentage of sperm in a population, which arecapable of responding to capacitating stimuli. In addition, incubationof the sperm under capacitating conditions and then adding a reagentwhich induces acrosomal exocytosis for example: progesterone,recombinant/solubilized or solid proteins from the zona pellucida (suchas the ZP3 protein in mouse or homologs from other species),carbohydrate moieties (such as the carbohydrate moieties on ZP3),calcium ionophore, or other pharmacologic agents (such aslysophosphatidylcholine) allows one to track the percentage of sperm ina population which are capable of responding to that stimulus.Therefore, the invention allows an individual (such as scientist,clinician, or herd health manager) to find out detailed informationregarding the ability of sperm to respond to different stimuli forcapacitation and acrosomal exocytosis.

Accordingly, in one embodiment, the invention provides a method fordetermining the pattern of G_(M1) distribution in non-capacitated spermin a species and determining the change in this pattern (redistribution)during capacitation. A control or standard pattern of G_(M1) stainingcan be obtained from a sample of normal semen. Typically, a controlpattern can be generated for a given species by evaluating the patternfrom a large sample (such as about 20–30) of individuals who have beenclassified as fertile according to acceptable criteria. In the case ofhumans, such criteria have been established by the World HealthOrganization (WHO). Further, the American College of Theriogenologypublishes guidelines for normal semen parameters for several species.The normal pattern of the redistribution of G_(M1) in response tocapacitating stimuli can also be determined for each species. G_(M1)staining can be observed for each sample to be tested in severalmicroscopic fields (typically 100–200 sperm). Various staining patternsare identified and a frequency chart is obtained for each pattern.Staining patterns that are most abundant in the normal samples aredesignated as normal staining patterns. It is expected that even in thenormal samples some deviations from the most abundant staining patternwill be observed. The number and frequency of such deviations can bedocumented to establish a range of frequencies for each type ofdeviation in normal semen. This will serve as a control or standardstaining pattern for comparison with test samples.

When evaluating a test sample, G_(M1) staining can be carried out asdescribed above and the staining pattern examined. The number andfrequency of each type of staining pattern is recorded and the data foreach test sample is compared to the control pattern. Samples may bedesignated as abnormal and suggestive of sub-fertility/infertilitybecause of staining patterns unique to the test samples (i.e., abnormallocalization as shown herein in FIG. 8) or on the basis of deviatedfrequencies of staining patterns from that observed in the controls.

In another application of this invention, the effect of variousenvironments (such as storage media, contraceptives or incubation mediaetc) or various protocols relating to storage, processing of semen orcontraception can be determined by assessing G_(M1) distribution. Thiswill serve as an indicator of the integrity of the sperm plasmamembrane, the integrity/organization of plasma membrane sub-domains, andthe ability of sperm to undergo capacitation and/or acrosomalexocytosis. For these applications, G_(M1) distribution patterns orlocalization following the exposure, storage or incubation of spermin/to various environments or protocols is compared to a controlpattern. The control pattern can be an established standard or may be agenerated from samples run in parallel with the test sample but withoutthe presence of the particular attribute being tested.

For example, G_(M1) distribution and its redistribution duringcapacitation is used for evaluation of sperm storage media such as semenextenders and cryopreservation media. In a variety of species (e.g.bovine, equine, porcine, ovine, canine, murine and human),cryopreservation and the use of semen extenders to ship cooled semen, orto wash/dilute fresh or previously frozen sperm, is an important part ofassisted reproduction and in the generation of transgenic animals. Inaddition, the freezing of sperm for long-term storage also requires theuse of agents and protocols that minimize damage to these cells. In mostcases, the semen extenders and agents used as protectants against damageduring freezing and thawing rely on poorly defined media components(e.g. milk or egg yolks). Optimization of such reagents and protocolscontinues to be of primary commercial importance, and the development ofa completely defined protective medium would give tremendous commercialadvantage regarding quality control. Because G_(M1) has been found onthe plasma membrane of sperm in discrete locations in sperm prior to andduring capacitation, and during acrosomal exocytosis, the localizationof G_(M1) can be used as a marker for how exposure to such reagents andprotocols affects the fragile organization of the membrane sub-domainsin these cells. In addition, because G_(M1) redistribution reflects asperm's response to capacitating stimuli and stimuli for acrosomalexocytosis (both requirements for the sperm to be able to fertilize anegg), the localization of G_(M1) can be used as a marker for howexposure to such reagents and protocols can affect the ability of thesperm to undergo capacitation and acrosomal exocytosis. Moreover,exogenous G_(M1) has been demonstrated to affect the signaling activityof a variety of cell types. We have found that commonly used commercialsemen extenders, often contain high amounts of exogenous G_(M1). Suchdata suggest that the appearance of this ganglioside in media might behaving pro- or anti-capacitation effects on the sperm, and might beaffecting the distribution of G_(M1) within the sperm.

To be useful in this regard, fresh semen can be collected, washed(typically accomplished by low centrifugation and resuspension in themedium or passing through cheesecloth to remove the gel fraction) andaliquots exposed to a candidate semen extender/cryopreservation mediaand protocols. Sperm from each group can be labeled with the marker forthe G_(M1) at various stages through this process, and either visualizeddirectly with epifluorescence, or incubated under capacitatingconditions and then visualized after washing.

Alternatively, sperm from each group could be fixed at various stagesthrough a process of cryopreservation or during incubations with stimulifor capacitation and/or acrosomal exocytosis, and then labeled, washed,and visualized. Comparison against a fresh ejaculate or an alreadyestablished standard can be carried out to determine if there has been aloss or gain of the ability to respond to capacitation and/or acrosomalexocytosis stimuli.

In another embodiment, G_(M1) distribution and its redistribution duringcapacitation and acrosomal exocytosis is used for evaluation of malefertility deficiencies. Idiopathic male infertility is an importantreproductive concern in a variety of species, including the human. Inagricultural and veterinary settings, breeding soundness exams are animportant component of evaluating male fertility. However, mostassessments short of fertility trials are based on gross observations ofsperm motility and morphology, as opposed to functional assays. BecauseG_(M1) is a marker for the organization of discrete plasma membranesub-domains, and because its redistribution reflects a response tocapacitating stimuli and stimuli for acrosomal exocytosis, thelocalization of this sub-domain in sperm can provide informationregarding both the organization of sperm at the ultra-structural level,and the ability of these cells to undergo changes associated with theacquisition of fertilizing ability. Therefore, an assessment of thelocalization of G_(M1) would provide detailed information not currentlyavailable. In addition, such a test could be performed within hours, asopposed to other tests which require at least two days to perform.

To be used as a diagnostic assay when assessing male fertility, spermcan be collected and washed according to standard protocols well knownin the art. They can then be labeled with the reagent specific forG_(M1), or they can be incubated under non-capacitating and capacitatingconditions (including stimuli for acrosomal exocytosis, if desired andthen labeled. Alternatively, they could be fixed and then labeled, orincubated under non-capacitating and capacitating conditions, fixed andthen labeled. The cells can be washed gently with the base medium andthen visualized by standard fluorescence microscopy. Because the assayrequires little time when compared with other methods of assessing spermfunction (e.g. hamster zona penetration tests or western blots to assessprotein tyrosine phosphorylation), knowledge gained from thisinvestigation could enable the physician or veterinarian to decidebetween several methods of assisted reproduction such asintra-cytoplasmic sperm injection (if the sperm were abnormal), orartificial insemination or in vitro fertilization (if the sperm showednormal patterns of G_(M1) prior to and upon exposure to capacitatingstimuli and stimuli for acrosomal exocytosis). Obtaining thisinformation for IVF would help to identify problem donors early in theprocess which can result in cost and time savings. Similarly,identification of problem males in species where breeding is routinelydone, can result in significant cost and time savings.

In another embodiment, G_(M1) distribution and its redistribution duringcapacitation and/or acrosomal exocytosis is used to predict future malereproductive fitness. In agricultural and veterinary settings, breedingsoundness exams are an important component of evaluating male fertility.Particularly in cattle, evaluation of the future reproductive success ofa given bull has significant economic impact. Initial assessments arebased on gross observations of sperm motility and morphology, as opposedto functional assays, and then are typically followed up over a periodof months and years with fertility trials. Because G_(M1) is a markerfor the organization of discrete plasma membrane sub-domains, andbecause its redistribution reflects a response to capacitating stimuliand acrosomal exocytosis, the localization of this sub-domain in spermcan provide information regarding both the organization of sperm at theultra-structural level, and the ability of these cells to undergochanges associated with the acquisition of fertilizing ability.Therefore, an assessment of the localization of G_(M1) would providedetailed information not currently available, potentially allowing herdmanagers to select for or against the continued use of a given male(bull in the case of cattle). In addition, such a test could beperformed within hours, as opposed to other tests which require at leasttwo days to perform.

To be used as a predictive indicator when assessing male fertility,sperm can be collected and washed according to standard protocols. Theycould be labeled immediately with the reagent specific for G_(M1), orthey could be incubated under non-capacitating and capacitatingconditions, and/or conditions promoting acrosomal exocytosis, and thenlabeled. Post-incubation, they can be fixed then labeled, or labeledthen fixed, or labeled as live cells, depending on the specificinformation desired. The cells can be washed gently and then visualizedby standard fluorescence microscopy. Because the assay requires littletime when compared with other methods of assessing sperm function (e.g.hamster zona penetration tests or western blots to assess proteintyrosine phosphorylation), knowledge gained from this investigationcould enable the herd manager or veterinarian to decide whether theindividual male had sperm which were normal with respect to theorganization of their plasma membrane sub-domains and in their responseto capacitating stimuli and/or stimuli for acrosomal exocytosis Becausecapacitation is essential for a sperm to fertilize an egg, a male havinga higher percentage of abnormal sperm than typical for its species couldbe selected to be removed from consideration for future breeding. Thiswould result in considerable economic savings.

In another embodiment, G_(M1) redistribution in response to capacitatingstimuli or stimuli for acrosomal exocytosis can be used for evaluationof potential contraceptives. Localization of G_(M1) in sperm can be usedto screen for the efficacy of male contraceptives, as well as a generaltool for assessment of environmental toxins on male reproduction. Theeffect of male contraceptives, including topical spermicides as well asparenteral and enteral pharmacologics, on sperm plasma membranes can beevaluated by using G_(M1) as a marker for the organization of discretesub-domains known to exist in normal sperm. Because the reagent specificfor G_(M1) can be detected by simple fluorescence microscopy, such atest could be performed quickly and conveniently in almost any researchsetting, or could be performed by a dedicated lab. Assessments of spermboth prior to and after incubation under capacitating conditions couldbe performed to look for subtle effects on fertilizing ability. Suchtests could be performed as described above. To establish a correlationbetween capacitation and G_(M1) pattern for a given species,capacitation can be established by standard methods such asimmunoblotting for protein tyrosine phosphorylation events.

The invention is further described through the examples presented belowwhich are intended to be illustrative and not restrictive in any way.

EXAMPLE 1

To localize the ganglioside, G_(M1), in living, motile sperm, 5×10⁶murine sperm were incubated with 10 μg/ml Cholera toxin b subunit withan Alexa-Fluor fluorescent tag (“CTb,” Molecular Probes, Eugene, Oreg.)in 750 μl of modified Whitten's medium (22 mM HEPES, 1.2 mM MgCl₂, 100mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic acid hemi-calciumsalt, pH 7.35). Glucose (5.5 mM), NaHCO₃ (10 mM ), and(2-hydroxypropyl)-β-cyclodextrin (2-OHCD; 3 mM) were supplemented asneeded for 10 minutes at 37° C. in the dark. After washing at 37° C.,CTb was seen specifically in the plasma membrane overlying the acrosome(FIG. 1). The fluorescence was detected with a Nikon TE2000 microscopeequipped with an OpenLab/Volocity imaging system (Improvision,Lexington, Mass.). The same pattern of fluorescence was seen when spermwere fixed with 4% paraformaldehyde and 0.1% glutaraldehyde.

EXAMPLE 2

To investigate the existence of lipid rafts in murine sperm, sterols,caveolin-1, and the ganglioside, G_(M1), were localized by fluorescencemethods. Caveolin-1 is often used as a marker for lipid rafts in that itrequires interaction with a sterol to attach to a membrane, and oftenfractionates with lipid raft membrane sub-domains. It should be notedthat caveolin cannot be detected in living cells by indirectimmunofluorescence because the epitopes recognized by the caveolinantibody are intracellular. Similarly, filipin must be visualized infixed cells as the autofluorescence is weak and easily quenched, hencemovement of the cells during a long exposure time would cause a loss ofresolution.

To localize sterols in murine sperm, we incubated 1×10⁶ sperm with0.005% filipin (w/v, in dimethyl formamide) (Sigma, St. Louis, Mo.) in 1ml of modified Whitten's medium (i.e. non-capacitating conditions).Sperm were washed twice by centrifugation and resuspended in mediumcontaining 0.004% paraformaldehyde (w/v). Filipin-sterol complexes (FSC)were visualized in sperm on a heated stage and chamber by theauto-fluorescence properties of filipin. FSC were seen in the acrosomalplasma membrane, the connecting piece, and the flagellum (FIG. 2A),which is consistent with published reports using freeze-fractureelectron microscopy. Caveolin-1 was localized using indirectimmuno-fluorescence (polyclonal antiserum #C13630, TransductionLaboratories, Lexington, Ky.), on cells permeabilized with methanol andfixed with 2% paraformaldehyde. Caveolin-1 was seen in the acrosomalplasma membrane, midpiece and principal piece (FIG. 2B). Control spermincubated with the secondary antibody alone were not labeled (data notshown). To localize G_(M1) in fixed sperm, 5×10⁶ murine sperm wereincubated with 10 μg/ml CTb in 750 μl of modified Whitten's medium for10 minutes at 37° C. in the dark. The sperm were fixed with 0.004%paraformaldehyde at 37° C., and then washed gently. Interestingly, CTbfluorescence was seen specifically in the post-acrosomal plasma membraneof the sperm head, the midpiece, the annulus, and a thin line down thelength of the principal piece (FIG. 2C). We have observed that themovement of G_(M1) from the plasma membrane overlying the acrosome tothe post-acrosomal plasma membrane can also occur upon cell death, solong as a cross-linking reagent (such as CTb or an anti-gangliosideantibody) is used, and the sperm had not been exposed to capacitatingstimuli (see below). Thus, the localization of G_(M1) in sperm dependsupon the response of the sperm to molecular stimuli for capacitation,the reagent used to visualize the G_(M1), and the fixation conditionused.

EXAMPLE 3

This example demonstrates the changes in the pattern of G_(M1)distribution (redistribution) that are observed under conditions whichare known to induce or which accompany the process of capacitation. InExample 2, it was demonstrated that the plasma membrane overlying theacrosome was enriched in sterols, whereas the post-acrosomal plasmamembrane was enriched in G_(M1), when the sperm were incubated undernon-capacitating conditions and then fixed in 0.004% paraformaldehyde.This segregation was remarkably consistent in non-capacitated sperm. Ithas been documented that exposure of sperm to sterol acceptors such as2-hydroxy-propyl cyclodextrin (2-OHCD) or bovine serum albumin (BSA)causes the loss of most FSC from the plasma membrane overlying theacrosome, and allows some FSC to diffuse laterally into thepost-acrosomal plasma membrane. This finding is consistent with the lossof sterols causing an increase in membrane fluidity, and promotinglateral diffusion. To investigate the dynamics of the rafts enriched inG_(M1), sperm were incubated as follows: under non-capacitatingconditions in a modified Whitten's medium containing Ca⁺⁺; with 10 mMHCO₃ ⁻; with 3 mM 2-OHCD; or with both 10 mM HCO₃ ⁻ and 3 mM 2-OHCD(capacitating). The last condition has been shown to cause the fullpattern of protein tyrosine phosphorylation associated withcapacitation, while leaving the sperm functional to perform in vitrofertilization (data not shown). Incubation with Ca⁺⁺, HCO₃ ⁻, or 2-OHCDalone is not sufficient to cause either tyrosine phosphorylation orcapacitation. Sperm were incubated for 45 minutes under theseconditions, then CTb was added for a 10 minute incubation at a finalconcentration of 10 μg/ml prior to washing. To sperms were then fixed in0.004% paraformaldehyde. Similar patterns were seen when sperm wereincubated under the various conditions, then fixed with 0.004%paraformaldehyde and then labeled with CTb.

FIG. 3A is an example of the most abundant different patterns seen undernon-capacitating and capacitating conditions. FIG. 4 shows the relativedistribution of these patterns under non-capacitating and capacitatingconditions. As can be seen in FIG. 4, the majority of sperm incubatedunder non-capacitating conditions had a type “A” pattern of labelingwith CTb. Incubation with HCO₃ ⁻ stimulated a shift from pattern “A” topattern “B.” Incubation with either 2-OHCD alone, or in conjunction withHCO₃ ⁻, caused almost identical shifts to pattern “C.” None of thetreatment conditions caused observable change in the localizationpatterns over the midpiece, annulus, or principal piece. It should alsobe noted that 45 minute incubations were chosen to minimize the percentof sperm that underwent spontaneous acrosomal exocytosis. Because G_(M1)localizes specifically to the plasma membrane, and the CTb used has aparticularly bright fluorescence, the integrity of the sperm membranes(and state of the acrosome) was clearly visible. Longer incubation timeswould be expected to show more pronounced shift to pattern C underconditions promoting sterol efflux. The following criteria for countingand assignment of a sperm into one of the patterns were used,including 1) counting only morphologically normal sperm labeled withequal intensity, 2) counting only sperm in areas having a uniformbackground signal, 3) assigning sperm based on the relative intensitiesof fluorescence in the acrosomal and post-acrosomal regions, and 4)counting all the sperm in multiple high power fields. The latter pointwas important to avoid investigators' sub-consciously counting aparticular pattern first, and then stopping when a count of 100 cellswas reached. To compare the shifts in population tendencies, the numberswere converted to percentages prior to graphing.

EXAMPLE 4

This example demonstrates patterns of G_(M1) localization in murinesperm after incubation under capacitating conditions followed byincubation in the presence of 10 μg/ml progesterone for 10 minutes.G_(M1) labeling was carried out as described above in Example 3. Theresults are shown in FIG. 5. Labeling of G_(M1) is seen concentrated inthe region of the apical acrosome. Both sperm were incubated wtihprogesterone. Both have signal over the apical acrosome, but not overthe entire acrosomal plasma membrane. The image on the left shows alinear region of clearing between two brighter lines on the curve of thesperm head. The one on the right shows less of a clearing, with thefluorescence more even over the apical acrosome.

EXAMPLE 5

This example demonstrates patterns of G_(M1) localization in bull spermand dog sperm. G_(M1) labeling was carried out as described in Example2, except using TALP (Tyrode's Albumin/Lactate/Pyruvate) medium for thebovine sperm (Parrish, et al., 1988, Biology of Reproduction,38:1171–1180), and canine capacitation medium for the dog (Mahi andYanagimachi, 1978, Gamete Research, 1:101–109). The results are shown inFIGS. 6 (bull) and 7 (dog). Several patterns of G_(M1) localization seenin a normal sample are shown. These patterns appear to be similar topatterns seen in the mouse.

EXAMPLE 6

FIGS. 8A and 8B demonstrate that G_(M1) distribution can be used as adiagnostic tool. To illustrate this embodiment, ejaculated sperm from ageriatric stallion having a defect of proximal droplets/pseudo-dropletswere used. It is clear that in this figure, the morphologically-obviousdefect in the proximal midpiece is accompanied by an apparent defect inlocalization of G_(M1) in the plasma membrane of the sperm head.Therefore, this invention can reveal defects in the organization of thesperm plasma membrane which are not detectable by ordinary lightmicroscopy.

While this invention has been described through illustrative examples,these examples are not intended to be limiting in any way and it will berecognized that routine modification can be made by those skilled in theart. Such modifications are intended to be included within the scope ofthe present invention.

1. A method of determining the responsiveness of sperm in a test sampleto one or more stimuli associated with capacitation and/or acrosomalexocytosis, comprising the steps of: a) determining a G_(M1)distribution pattern for sperm in the test sample, comprising: (i)exposing the test sperm to the one or more stimuli; (ii) fixing the testsperm in a fixative comprising paraformaldehyde, glutaraldehyde orcombinations thereof; (iii) staining the fixed test sperm to determinethe G_(M1) distribution pattern in the fixed test sperm; b) obtaining aG_(M1) distribution pattern of a normal sperm sample which has beenexposed to the same one or more stimuli and fixed with the same fixativeas the test sperm; c) comparing the G_(M1) distribution patterns of thefixed test sperm and the fixed normal sperm, wherein a differencebetween the G_(MI) distribution patterns of the fixed test sperm and thefixed normal sperm is indicative of an abnormal capacitation and/oracrosomal exocytosis response of the test sperm to the one or morestimuli.
 2. The method of claim 1, wherein the difference between theG_(M1) distribution patterns is an abnormal localization of G_(M1)within the fixed test sperm compared to the fixed normal sperm.
 3. Themethod of claim 1, wherein the difference between the G_(M1)distribution patterns is a difference in the relative frequencies ofdifferent G_(M1) distribution patterns in the fixed test sperm comparedto the frequencies of the same patterns in the fixed normal sperm. 4.The method of claim 1, wherein the one or more stimuli associated withcapacitation is selected from the group consisting of calcium,bicarbonate, a mediator of sterol efflux, and combinations thereof. 5.The method of claim 4, wherein the mediator of sterol efflux is selectedfrom the group consisting of 2-hydroxypropyl-β-cylcodextrin,methyl-β-cyclodextrin, serum albumin, high density lipoprotein,phospholipid vesicles, liposomes and combinations thereof.
 6. The methodof claim 1, wherein the one or more stimuli associated with acrosomalexocytosis is selected from the group consisting of progesterone,calcium ionophore, zona pellucida proteins, lysophosphatidylcholine, andcombinations thereof.
 7. The method of claim 1, wherein the test sampleis from an individual selected from the group consisting of cat, cattle,dog, goat, guinea pig, horse, human, mouse, pig, rabbit, rat and sheep.8. The method of claim 1, wherein the fixative comprises 0.004%paraformaldehyde.
 9. The method of claim 1, wherein the fixativecomprises 4% paraformaldehyde, 0.1% glutaraldehyde and 1mM CaCl₂. 10.The method of claim 1, wherein the fixative comprises 1.25%paraformaldehyde and 2.5% glutaraldehyde.
 11. The method of claim 10,wherein the fixative further comprises 100 nM sodium cacodylate and 0.5mM CaCl₂.